The Epstein-Barr virus (a γ herpes virus that stays dormant (latent) for a long period in memory B cells; hereinafter may be abbreviated as EBV) is involved in many malignancies, for example, Burkitt's lymphoma, Hodgkin's disease (HD) and nasopharyngeal carcinoma (NPC), as well as post-transplant lymphoproliferative disorder (Non-patent Document 1). In latent infections, viral protein expression is suppressed. All EBV-positive malignant cells exhibit one of the following three latency types. These types are distinguished from each other by the EBV antigen expression patterns (Non-patent Document 2).
Latency type I: only EBV nuclear antigen (EBNA) 1 is expressed in Burkitt's lymphoma.
Latency type II: latent membrane protein 1 (hereinafter abbreviated as LMP1) and LMP2, as well as EBNA1, are expressed in Hodgkin's disease and nasopharyngeal carcinoma.
Latency type III: all EBV latent proteins, i.e., EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, leader protein, LMP1, and LMP2 are expressed in post-transplant lymphoproliferative disorder.
There is increasing interest in using immunotherapy for EBV-associated malignancies. Adoptive immunotherapy using in vitro-activated EBV-specific cytotoxic T lymphocytes (hereinafter may be abbreviated as CTLs) has proven effective for prevention and treatment of EBV-associated lymphoproliferative disorders following hematopoietic stem cell or organ transplantation (Non-patent Documents 3-7). Application of a similar strategy to other EBV-associated malignancies such as Hodgkin's disease (Non-patent Document 8) and nasopharyngeal carcinoma (Non-patent Document 9) has been reported to be effective in some patients. However, the majority of lymphoblastoid cell line (hereinafter abbreviated as LCL)-activated CTLs used in these studies were directed to EBNA3A, EBNA3B, and EBNA3C, which antigens are not expressed in malignant tumors such as Hodgkin's disease and nasopharyngeal carcinoma. A portion of LCL-activated CTLs might recognize peptides derived from LMP2 and contribute to immunotherapeutic effects in patients (Non-patent Documents 8-9). However, T cells directed to LMP1 peptides are very rare, reflecting low frequency of LMP1 peptide-specific CTL precursor cells (Non-patent Document 10). To selectively activate T cells specific to subdominant EBV antigens, Lin et al. used monocyte-derived dendritic cells (DCs) pulsed with LMP2-derived peptides to immunize NPC patients (Non-patent Document 11). Some methods for activating LMP1-specific CTLs have also been reported. Khanna et al. first described HLA-A2-restricted LMP1 epitopes and induction of CTLs using antigen-presenting cells (APCs) pulsed with the peptides (Non-patent Document 10). They also utilized a replication-incompetent adenovirus and a recombinant vaccinia virus encoding multiple LMP1 epitopes, successfully immunizing HLA-A2 transgenic mice, and achieving inhibition of the growth of LMP1 gene-transduced cells (Non-patent Documents 12-13). Gottschalk et al. reported the induction of polyclonal LMP1-specific CTLs using dendritic cells infected with a recombinant adenovirus expressing an N-terminal-truncated, nontoxic LMP1 mutant (Non-patent Document 14).
One category of EBV-associated malignancies is EBV infection in NK/T cells (Non-patent Document 1). Chronic active EBV infection (CAEBV) is another disorder whereby EBV infects mainly NK/T cells to cause life-threatening lymphoproliferative disease (Non-patent Document 15). EBV-positive NK/T cell malignancies express EBNA1 and LMP1 as potential CTL targets (Non-patent Documents 15-17).
LMP1 is a transmembrane oncoprotein that enhances cell survival through upregulation of anti-apoptotic genes (Non-patent Document 2). Expression of LMP1 is essential for growth transformation of human B-lymphocytes and is necessary for the proliferation of EBV-infected human monocytes (Non-patent Document 2). LMP1 has also been known to induce tumorigenic transformation of the murine cell line BALB/c3T3 and B cell lymphomas. Moreover, LMP1 expression might be important for the proliferative capacity of EBV-infected NK cells (Non-patent Document 18). However, it has not been demonstrated that such NK/T cells can process LMP1 and generate HLA-restricted epitopes.
EBNA1 is required for the maintenance and replication of viral plasmids in EBV-transformed cells (Non-patent Document 19). Because EBNA1 is expressed in all EBV-associated tumors, it is an attractive target for immunotherapy. However, CD8+ CTL responses are preferentially directed toward EBNA3A, EBNA3B, and EBNA3C among latent antigens, and EBNA1 has been believed to be not recognized by CTLs and thus to be immunologically undetectable (Non-patent Documents 20-23). A glycine-alanine repeat sequence (GAr) within EBNA1 was found to prevent antigen processing for CTL recognition (Non-patent Document 24). Presence of this GAr proved to prevent processing by proteasomes, the main catalytic machinery for generating MHC class I epitopes (Non-patent Documents 25-26). Moreover, the very same domain was found to prevent EBNA1 mRNA translation (Non-patent Document 27).
EBV-specific CD4+ T cell responses have been examined and it has been elucidated that EBNA 1-specific CD4+ T cell responses are seen mainly in helper T cell type 1 that directly recognizes EBV-infected cells. Several MHC class II-restricted EBNA1 epitopes have been identified (Non-patent Documents 28-32), implying that EBNA1-specific CD4+ T cells may play a role in controlling tumor growth in vivo. Surprisingly, very recent studies demonstrated EBNA1-specific CD8+ CTLs to moderately lyse EBV-infected lymphoblastoid cell lines (LCLs) and suppress LCL outgrowth in vivo (Non-patent Documents 33-35). However, only a small number of EBNA1 epitopes that activate CD8+ CTLs have been identified so far.
Induction of CTLs with very few precursor cells has been tried by immunologists as well as clinicians anticipating tumor-associated antigen-targeted immunotherapy. Antigen-presenting cells transduced with in vitro-transcribed mRNA encoding specific antigens can induce CTLs specific to tumor-associated antigens, or to self antigens, overcoming immunological tolerance (Non-patent Documents 36-40). The above methods have the following advantages:    (1) complete loss of antigenicity of vector backbone sequences;    (2) highly reproducible yields with in vitro transcription; and    (3) high efficiency of transduction using electroporation.
Namely, it is inferred that antigen-presenting cells transduced with mRNA of EBV antigens (epitopes) might be suitable for induction of EBV-specific CTLs. To realize the above methods, identification of CTL epitope peptides specific to EBV was needed.
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